GSM6022396: SI Crypts_KO3 [Bisulfite-seq]; Mus musculus; Bisulfite-Seq
We followed a previously published protocol to isolate intestinal crypts [PMID: 23097115]. Briefly, the small intestine (Jejunum) was flushed with ice-cold PBS then cut open longitudinally. The Jejunum was minced into small pieces in cold PBS (2-5 mm) and then incubated for 30 min at 4 °C in PBS containing 2 mM EDTA. Intestinal fragments were then transferred into cold PBS and the villi fraction was mechanically isolated by shaking. Then the intestinal fragments were transferred into fresh cold PBS and the crypts fraction was mechanically isolated by vigorously shaking. This step was repeated further two times to recover the crypts. Crypts fractions were then combined and passed through a 70 µm cell strainer. The combined fractions of crypts were then washed in PBS, centrifuged at 300g for 3 min, and stored at -80°C for DNA extraction. Genomic DNA was extracted from intestinal epithelial crypts using DNA blood and tissue kit (cat. no. 69504, Qiagen), according to the manufacturer's instructions. Accel-NGS Methyl-Seq DNA